| ObjectInsomnia is a common clinical sleep disorder.As many as 10%-20%of adults meet the diagnostic criteria for insomnia.Most of them show chronic progress and are positively correlated with age.At the same time,insomnia can induce mental disorders,cardiovascular and cerebrovascular diseases,nervous system diseases,endocrine and immune diseases,etc.,which seriously affect the physical and mental health of patients and affect the quality of life of patients.Sedatives,hypnotics,anti-anxiety,antidepressants,and hormone replacement therapy are often used for insomnia,but the adverse reactions that occur during the medication process have always been the difficulties faced by this type of treatment drugs.The treatment of insomnia in traditional Chinese medicine is also based on the dynamic changes of the body's yin and yang in the circadian rhythm.It not only pays attention to the quality of night sleep,but also regulates the state of energy during the day and restores the biological rhythm characteristics of the body's circadian essence.Guizhi Gancao Longgumuli Decoction refers to a formula derived from Guizhi Gancao Longgumuli Decoction.Chaihu plus Longgumuli Decoction,Sini Decoction,Guizhi Gancao Longgumuli Decoction,Guizhi plus Longgumuli Decoction are all classic prescriptions for the treatment of insomnia,and they all use Guizhi Gancao Longgumuli Decoction as the basic prescription,but clinically treat insomnia The types and symptoms vaiy.Therefore,this study used the zebrafish model biotechnology platform to study the effects of Guizhi Gancao Longgumuli Decoction and its analogues on zebrafish sleep circadian rhythms,and through the comparative analysis of zebrafish pharmacodynamic fingerprints,to explore Guizhi The influence of Zhigancaolonggumuli decoction on zebrafish circadian sleep phase parameters and possible pathways and effect targets pro vide theoretical basis for prescription optimization and new drug discovery,and explore new ways of traditional Chinese medicine compound and single-drug research.MethodsExperiment 1:To establish a zebrafish insomnia model using drug-induced methods,select the drugs 17?trenbolone and caffeine to soak 5dpf zebrafish for 48 hours respectively,observe the morphological development of the zebrafish,and evaluate the zebrafish after drug-induced behavior.Fluorescence quantitative PCR was used to determine the expression level of zebrafish sleep-related genes,and the best insomnia model drugs were selected through comprehensive evaluation.Experiment 2:The zebrafish insomnia model was established by drug induction,and the zebrafish insomnia model juveniles treated with each single agent of Guizhi Gancao Longgu Muli Decoction were subjected to continuous 48 hours of sleep behavior testing using the zebrafish behavior detection system.Analyze six behavioral indicators related to sleep/wakeness(total sleep time,number of sleep cycles,length of sleep cycle,sleep latency,total activity,and wakeful activity),and construct a fingerprint of the efficacy of each single drug for visualization deal with.The total sleep time and the amount of wakeful activity,which are representative parameters that reflect sleep and wakeful behavior,were analyzed in the sleep/wake time series.Experiment 3:Prepare a zebrafish insomnia model,and use the zebrafish behavior detection system to test the sleep behavior of zebrafish juveniles treated with Guizhi Gancao Longgu Oyster Decoction for 48 hours,and observe the day and night at the same time The spontaneous behavior of zebrafish analyzes the six behavioral indicators related to sleep/wakeness,constructs the behavioral fingerprint of Guizhi Gancao Longgu Oyster Decoction,and visualizes it.Through time series analysis,cluster analysis,correlation analysis and other data processing methods,it shows the zebrafish's behavioral phenotype,clustering law,and the strength of correlation.The correlation analysis between the behavioral fingerprints induced by Guizhi Gancao Longgumuli Decoction and the compound database established by published articles was carried out,the Pearson correlation coefficient was calculated,and the candidate neural signal pathways were screened out according to the degree of correlation.Finally,real-time fluorescent quantitative PCR was used to detect the effect of Guizhi Gancao Longgumuli Decoction on the mRNA expression levels of genes related to the prediction pathway of the zebrafish insomnia model,and the effect and prediction of Guizhi Gancao Longgumuli Decoction were verified at the molecular level.Correlation of pathways.Results1.Experimental result one:(1)Starting from the lowest concentration of lOng/L group,17?-trenbolone immersed 4hpf zebrafish embryos to develop to 4dpf,which had a significant impact,and the malformation rate exceeded 50%;while for 5dpf zebrafish juveniles at each concentration of 17?-trenbolone Within 48 hours of soaking in the solution environment,no obvious changes were seen,suggesting that induction of 17?-trenbolone during the embryonic stage of zebrafish may affect the growth and development of zebrafish.(2)In the spontaneous learning experiment,compared with the blank group,17?-trenbolone significantly reduced the total movement distance of zebrafish juveniles by 5dpf(P<0.05),and the activity speed slowed down(P<0.05),suggesting that the juveniles were exposed In the environment of 17?-trenbolone,it can also affect its motor behavior.17?-trenbolone may affect the motor neurons of zebrafish juveniles,which is consistent with the results of the literature.Compared with the blank group,zebrafish juveniles in the caffeine group showed a significant increase in the total distance,total distance and average speed of movement,and showed a more active state(P<0.05).(3)Fluorescence quantitative PCR was used to detect zebrafish sleep-related genes and found that compared with the blank group,the per1a mRNA expression level of 5dpf juveniles exposed to 17?-trenbolone was significantly up-regulated(P<0.05),while the biological species genes bmal1a,bmal1a,There was no significant change in the mRNA expression of per2 and 5-HT transport protein sert(P>0.05).Compared with the blank group,the expression level of the circadian clock gene bmal1a mRNA in the caffeine group was significantly down-regulated(P<0.05),and the expression level of the circadian clock genes per la,per1b,per2,and per3 showed an upward trend,suggesting that caffeine has an effect on zebrafish.Changes in sleep behavior may affect the expression of per1a,per1b,per2,per3 and other circadian clock genes;while the mRNA expression of species gene bmal1a did not change significantly(P>0.05).It is suggested that caffeine affects the normal regulation of the zebrafish circadian clock feedback loop.The drug caffeine can successfully induce the zebrafish insomnia model.The modelling effect is more stable than that of 17?-trenbolone,and it can show anxiety-like behavior at the same time.2.Experimental result two:(1)Compared with the insomnia model group,the Guizhi high,medium,and concentration groups can increase the amount of daytime awakening activity and total daytime activity of zebrafish juveniles,increase the total night sleep time and reduce the total day sleep time The number of rounds during the day.At the same time,high,medium,and low concentrations of Guizhi can reduce the length of sleep rounds of juvenile fish during the day and night,as well as the sleep latency at night.For the daytime sleep latency,it increased in the high and medium concentration group of Guizhi,and decreased in the low concentration group.In terms of nighttime awakening activity,the high and medium concentration group of Guizhi increased and decreased in the low concentration group.Compared with the insomnia model group,the licorice high,medium,and low concentration groups can increase the total night sleep time,the number of night sleep,the total amount of activity during the day and night,the amount of awakening activity during the day,and the sleep latency during the day.Reduce the length of the daytime sleep cycle,and the nighttime sleep cycle length is slightly reduced;as for the total daytime sleep time,the high and low concentration groups show a decrease,and the medium concentration shows a slight increase;the nighttime wakefulness activity is medium,high and low concentrations The group showed a decrease,and the medium concentration showed an increase.Compared with the insomnia model,the keel high and medium concentration groups generally increased the total sleep time of the juveniles during the day and night,the number of sleep during the day and night,the total amount of activity during the day and night,and the awakening activities during the day and night.The total amount of sleep during the day and night was reduced;the keel low concentration group also increased the total activity and wakefulness of the juveniles during the day and night,but the total sleep time during the day was expressed as a decrease.Compared with the insomnia model,the oyster high,medium,and low concentration groups can increase the total activity of juveniles during the day and night,the activity of awakening,the number of night sleep rounds,and the sleep latency during the day,and reduce or slightly reduce the day.The total sleep time between days,the length of day and day sleep cycles;and the night sleep latency,the oyster high and medium concentration group showed a slight increase,while the low concentration group showed a slight decrease.(2)The results of time series analysis showed that compared with the insomnia model group,the Guizhi group had no significant changes in the total time of day and night sleep at low,medium and high concentrations,and the main reason was to increase the amount of daytime wakefulness activity.The results of the licorice high,medium,and low concentration groups were similar to those of the Guizhi group.There was no significant difference in the total sleep time during the day and night with the model group,but it increased the amount of awakening activity during the day.The keel group showed that it increased the total sleep time,while increasing the amount of daytime awakening activity to a certain extent.The low,medium and high concentration group of the oyster group did not significantly change the total sleep time,but increased the amount of daytime wakefulness activity to a certain extent.The positive drug melatonin group can increase the total sleep time and reduce the amount of daytime wakefulness activity,which is manifested as the effect of inhibiting zebrafish vitality.3.Experimental result three:(1)Efficacy fingerprint of Guizhi Gancao Longgumuli Decoction:Compared with the model group,Chaihu plus Longgumuli Decoction mainly showed that it increased the total day and night sleep time,the length of sleep rounds,the total amount of activity at night,and day awakening.The amount of activity in the medium and low concentration groups has obvious effects,while reducing the number of day and night sleep cycles,and the total amount of daytime activity is the amount of awakening activity at night;Sini Decoction and Guizhi Gancao Longgu Muli Decoction are mainly manifested by increasing day and night The amount of awakening activity increased during the day and night sleep latency,but reduced the total daytime sleep time and total daytime activity;compared with the model group,Guizhi Gancao Longgu Muli Decoction can increase the total night sleep time,daytime The number of sleep rounds,the length of sleep rounds during the day and night,the sleep latency during the day and night,and the amount of awakening activity during the day and night,but the total time of sleep during the day and the total activity during the day and night showed a decrease;with the model group In comparison,Guizhi plus Longgu Muli Decoction can increase the total day and night sleep time,the length of sleep rounds,and the amount of awakening activity during the day,while reducing the number of sleep rounds and the amount of awakening activity at night.(2)Time series analysis results show that compared with the model group,the low-concentration group of Chaihu plus Longgumuli Decoction can increase the amount of zebrafish's daytime awakening activity,but the change in total sleep time is not obvious.The medium and high-concentration groups are respectively It increases the total sleep time of zebrafish and reduces the amount of wakefulness during the day.Compared with the model group,the low-concentration group of Sini Decoction and Guizhi Gancao Longgumuli Decoction had no significant changes in total sleep time,but there was a slight increase in the amount of daytime arousal activity,while the medium and high-concentration groups could increase the number of days.For the total rest time,the effect of medium concentration is the most obvious.Compared with the model group,the Guizhi Gancao Longgumuli Decoction low,medium,and high concentration groups all increased the amount of daytime wakefulness activity,while the medium and high concentration groups increased the total sleep time.Compared with the model group,the low,medium,and high concentration groups of the Guizhi plus Longgumuli Decoction group can increase the total sleep time,while the low and medium concentrations also increase the amount of wakefulness during the day.The amount of arousal activity does not change significantly.Compared with the model group,the positive drug melatonin group increased the total sleep time,and at the same time reduced the amount of daytime awakening activity,showing the effect of inhibiting zebrafish activity.Compared with the sleeping drug melatonin,in addition to improving sleep,Guizhi Gancao Longgu Muli Decoction can also maintain the circadian rhythm of zebrafish sleep and wake behavior.(3)In the spontaneous learning experiment,compared with the insomnia model group,the low concentration of the Chaihu plus Longgumuli Decoction group increased the total distance of movement during the day(P<0.05),and with the increase of the drug concentration,the middle concentration and the The total moving distance of the high concentration group is reduced.Compared with the insomnia model group,there is no statistical difference between the concentration groups of Sini Decoction and Guizhi Gancao Longgu Muli Decoction,but as the concentration increases,the total moving distance shows an increasing trend;compared with the insomnia model group,Guizhi Gancao Longgu The concentration in the Muli Decoction group can increase the total distance of movement during the day.Compared with the melatonin group and the high and low concentration groups,the difference is statistically significant.Compared with the insomnia model group,the Guizhi plus Longgumuli Decoction group has There was no significant change in the total travel distance.Compared with the insomnia model group,the low concentration of each concentration group of Chaihu plus Longgumuli Decoction increased the average daily moving speed,and with the increase of the drug concentration,the moving speed slowed down;compared with the insomnia model group,Sini Decoction combined with Gui Although there is no statistical difference between the concentration groups of Zhigancao Longgumuli Decoction,as the concentration increases,the moving speed shows an increasing trend;compared with the insomnia model group,the moving speed of the zebrafish in the concentration group of Guizhi Gancao Longgumuli Decoction It showed a significant increase,but there was no significant change in the high and low concentration groups;compared with the melatonin group and the high and low concentration groups,the differences were statistically significant;compared with the insomnia model group,the Guizhi plus Longgumuli Decoction group was within 30 minutes The speed of movement has not changed significantly.Compared with the model group,the total moving distance and average moving speed of each group of zebrafish in 30 minutes at night showed no significant difference.(4)Correlation analysis between the class prescription groups showed that the correlation between the high,middle and low concentration group of Chaihu plus Longgumuli Decoction and the concentration groups of other class prescriptions>0.7 are:5,2,6;Sini Decoction high,medium and low concentration The frequency of the correlation between each concentration of the group and other prescriptions>0.7 is:4,5,0;the frequency of the correlation between the concentration of Guizhi Gancao Longgumuli Decoction group and other prescriptions>0.7 is:3,6,6;The frequency of the correlation between the concentration of Guizhi plus Longgumuli Decoction in the high,middle and low concentration group and other prescriptions>0.7 is:3,7,6.The results with the highest correlation frequency within each class group are:Chaihu plus Longgumuli Decoction Low Concentration Group(CHL),Sini Decoction and Guizhi Gancao Longgumuli Decoction Medium Concentration Group(SNM),Guizhi Gancao Longgumuli Decoction Medium Concentration Group(SNM),Guizhi Gancao Longgumuli Decoction Low concentration group(GGL),Guizhi plus Longgumuli decoction medium concentration group(GZM),the above four treatment groups are the concentration treatment groups with the highest similarity of Guizhi Gancao Longgumuli decoction on zebrafish behavioral phenotypes.The results are consistent with the results of the K cluster analysis between the class squares.(5)The correlation analysis of the drug concentration in the class prescription group showed that there was a high degree of correlation between the high and low concentration groups in the Chaihu plus Longgumuli Decoction group(the correlation coefficient between the groups was greater than 0.9).and the Sini Decoction and Guizhi Gancao Longgumuli There is a high degree of correlation between the high and medium concentration groups of soup(the correlation coefficient between groups>0.8),and there is a high degree of correlation between the high and medium concentration groups of Guizhi plus Longgumuli Decoction,and between the medium and low concentration groups(between groups).Correlation coefficients are all>0.75)(6)Correlation analysis of the behavioral fingerprints of the four prescriptions and the database of the comparison range found that there are 48 similar drugs involved in the Guizhi Gancao Longgumuli Tang prescriptions,and the nerve compounds involved are mainly adrenal glands.Energy.5-HT energy,dopaminergic,GABAergic,melatonergic,histaminergic,adenylatergic,glutamatergic.In addition to Sini Decoction and Guizhi Gancao Longgumuli Decoction,the Chaihu plus Longgumuli Decoction group,Guizhi Gancao Longgumuli Decoction group,and Guizhi plus Longgumuli Decoction group showed different concentrations to regulate 5-HT energy and dopaminergic energy.The compounds in the system are highly correlated,and among them,the correlation with 5-HT compounds is the most frequent.(7)The real-time fluorescent quantitative PCR results showed that compared with the model group,the expression of sert mRNA in zebrafish juveniles in the four groups did not show significant statistical differences.The sert mRNA of zebrafish juveniles in the Sini Decoction and Guizhi Gancao Longgumuli Decoction group showed a downward-regulated trend with the increase of the drug concentration.The other groups did not show a significant dose-effect relationship with the increase of the concentration.Compared with the model group,5-H1A mRNA in the low-concentration group of Chaihu plus Longgumuli Decoction was significantly up-regulated(P<0.01),and the middle-concentration group was significantly down-regulated(P<0.01).Compared with the model group,Sini Decoction and Guizhi Gancao Longgu The 5-H1A mRNA in the low and medium concentration groups of Muli Decoction were significantly down-regulated(P<0.01).Compared with the model group,the 5-H1A mRNA in each concentration group of Guizhi Gancao Longgumuli Decoction showed significant differences,while the low concentration group showed significant differences.It was significantly up-regulated(P<0.01),and the medium and high concentration groups were significantly down-regulated(P<0.01).Compared with the model group,the 5-H1A mRNA in the Guizhi plus Longgumuli Decoction group showed significant differences,with low concentration,medium and high concentration.The concentration group showed a significant down-regulation(P<0.01),and as the drug concentration increased,the lower the down-regulation level was.Conclusion1.Guizhi Gancao Longgu Muli Decoction The Guizhi and Licorice are mainly used to promote daytime awakening activity,while Longgu and Oyster are mainly used to increase the total sleep time and jointly play a role in improving the sleep of the zebrafish insomnia model.2.Guizhi Gancao Longgumuli Decoction and its analogues can increase the amount of daytime awakening activity in the zebrafish insomnia model,and increase the total sleep time at the same time.Among them,the effect of Chaihu plus Longgumuli Decoction on the sleep/wake behavior of zebrafish insomnia It exhibits two-way regulation of low-concentration excitement and high-concentration inhibition.Sini Decoction and Guizhi Gancao Longgu Muli Decoction have more obvious excitatory effects on zebrafish3.The caffeine-induced zebrafish insomnia model is more in line with clinical insomnia with anxiety symptoms.The two-way regulating effect of Chaihu plus Longgumuli Decoction is better for its treatment effect.4.Sini Decoction and Guizhi Gancao Longgumuli Decoction may mainly act by mediating GABAergic signaling pathways.Chaihu plus Longgumuli Decoction,Guizhi Gancao Longgumuli Decoction and Guizhi plus Longgumuli Decoction may mediate 5-HT Energy and dopaminergic signaling pathways play a role. |
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